Synthesis and characterization of fluorescent probes targeting Histone Deacetylase 6 for live cell imaging

by Van Thang Nguyen
Doctoral thesis
Date of Examination:2025-11-05
Date of issue:2025-12-10
Advisor:Dr. Grazvydas Lukinavicius
Referee:Dr. Grazvydas Lukinavicius
Referee:Prof. Dr. Argyris Papantonis
Persistent Address: http://dx.doi.org/10.53846/goediss-11688

 

 

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Abstract

English

Histone Deacetylase 6 (HDAC6) is a unique enzyme that plays a central role in major cellular processes, including cytoskeletal regulation, protein quality control, and stress responses. Its dysregulation is linked to many diseases, from cancer to neurodegeneration, making it a critical therapeutic target. However, studying the dynamic functions of HDAC6 in its native cellular environment has been hampered by a lack of specific chemical tools. The development of selective, live-cell compatible fluorescent probes is essential for visualizing HDAC6 and elucidating its complex biological roles in real-time. This thesis reports the rational design, synthesis, and characterization of a novel, high-performance fluorescent probe for imaging HDAC6 in living cells. Employing a modular design strategy, a library of probes was synthesized based on established HDAC6 inhibitor. A robust, cell-based screening platform was developed, utilizing a panel of U-2 OS cell lines engineered to express individual Halo-tagged HDACs (HDAC1-8). This platform enabled the quantitative evaluation of probe selectivity via co-localization analysis. Through this high-throughput screening, the Nexturastat A scaffold was identified as a superior recognition motif, exhibiting exceptional selectivity for HDAC6. A subsequent, rigorous structure-activity relationship (SAR) study was performed to optimize the probe's performance. This systematic optimization led to the development of 6SiR-C3-NextA, which incorporates a bright, photostable, and far-red silicon-rhodamine (SiR) fluorophore, a C3 alkyl linker, and a hydroxamic acid zinc-binding group. Comprehensive characterization demonstrated that 6SiR-C3-NextA binds to HDAC6 with high affinity (Kd = 24 nM) and excellent selectivity. Crucially, the probe was found to be highly suitable for live-cell imaging, exhibiting minimal cytotoxicity and no significant perturbation of the cell cycle at effective imaging concentrations. The probe successfully visualized endogenous HDAC6 in a diverse panel of cell lines. Its on-target specificity was validated through competitive inhibition assays and co-localization with a specific anti-HDAC6 antibody. The utility of 6SiR-C3-NextA was further demonstrated in biological investigations, facilitating high-resolution imaging of HDAC6's association with the microtubule network and its dynamic recruitment to stress granules following osmotic shock. While the probe performed exceptionally well in most cell types, its application in primary neurons suggested some off-target interactions, highlighting an area for future refinement.
Keywords: histone deacetylases; fluorescent probes; fluorescence microscopy; live-cell imaging